Feasibility of expanding ischemia time for hearts destined for transplantation

延长移植心脏缺血时间的可行性

• 批准号: 10082625
• 负责人: Kelvin G.M. Brockbank
• 金额: $ 38.6万
• 依托单位: TISSUE TESTING TECHNOLOGIES, LLC
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-23 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10082625
• 关键词: Air; AlamarBlue; Amino Acids; Animal Model; Animals; Anions; Antioxidants; Apoptosis; Apoptosis Inhibitor; Biological Assay; Blood; Blood Vessels; Body Fluids; Capital; Cardiac; Cardiac Myocytes; Cell Line; Cell Survival; Cell membrane; Cells; Charge; Chloride Ion; Clinical; Collaborations; Complement; Consumption; Cryopreservation; Culture Media; Cyclic GMP; Data; Development; Enhancers; Environment; Equilibrium; Evaluation; Extracellular Space; Family suidae; Formulation; Gluconates; Gold; Grant; Heart; Heart Transplantation; Heterotopic Transplantation; Histopathology; Hour; Human; Hydrogen Sulfide; Ice; In Vitro; Inbred BALB C Mice; Intracellular Space; Ions; Ischemia; Isotonic Solutions; Lead; Letters; Lipids; Liver; Lobe; Mammals; Metabolic; Metabolism; Methodology; Methods; Molecular; Mus; Organ; Organ Donor; Organ Procurements; Organ Transplantation; Osmolalities; Outcome; Patients; Phase; Physiological; Plasma; Post-Translational Protein Processing; Postoperative Period; Potassium; Production; Proteomics; Pump; Reagent; Research; Safety; Saline; Ships; Small Business Innovation Research Grant; Sodium; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stains; Swelling; Technology; Testing; Time; Tissue imaging; Tissues; Transplantation; Trypan Blue; United Network for Organ Sharing; United States; Universities; Waiting Lists; Water; Work; World Health Organization; base; clinical practice; design; efficacy evaluation; experience; extracellular; glucose analog; heart function; heart preservation; improved; in vitro Assay; in vivo; inflammatory marker; innovation; macromolecule; meetings; metabolic rate; natural hypothermia; oxidation; post-transplant; preservation; pressure; scale up; screening; stress tolerance; tissue culture; transplant centers; transplant model; web site

In collaboration with Prof. Brandacher at Johns Hopkins University we plan to improve the currently most used clinical method of heart preservation, namely hypothermia at 0 to +4°C. We will build upon our base preservation formulation, Unisol™, that has been shown to preserved whole large animals below +10°C for 6- 8h after total blood replacement with Unisol™ with normal functions upon return to physiological conditions. We have also shown that Unisol™ can maintain blood vessel function for at least 6 days at both -7° C and +4°C and that mouse hearts stored at +4°C for 18 hours in Unisol™ have a significantly faster return of heart function than hearts stored in the gold standard hypothermic heart preservation solution Celsior (HTK). Encouraged by these results we propose evaluation of Unisol supplemented with reagents targeting oxidation, apoptosis (enhancers of stress tolerance) and metabolism (metabolic rate inhibition) in 2 specific aims using a human cardiac myocyte cell line to select optimal reagent concentrations in vitro and a heterotopic heart transplant model to evaluate the best supplement formulations developed in the in vitro studies. The lead in vitro assay will be alamarBlue, however outcomes will be checked using alternative assays including trypan blue, live/dead stain and MTT assay. Apoptosis will be evaluated if significant losses of metabolic activity are observed 1 to 2 days after return to physiologic culture conditions. MALDI tissue imaging and proteomics will be performed on selected control and best performing hearts to provide molecular mapping information on metabolites, lipids, enzymatic products, and post translational modifications in heart sections. The in vivo studies will evaluate the best formulations from the in vitro studies over 7 days of hypothermic storage using a syngeneic BALB/C mouse, heterotopic transplantation model. Controls hearts will be preserved in high K+ Unisol™ (UHK) and HTK. We anticipate that the supplemented formulation will maintain viability and function of hearts for at least 36 hours, a considerable improvement over all current practice methods. Based upon preliminary data with blood vessel preservation, heart storage times of 6-7 days may be obtained. This innovation will not only increase storage time, it will also provide the opportunity for more closely matched recipients and potentially induction of tolerance. Furthermore, we are improving on the most tried and true method for hypothermic heart storage in clinical practice that is relatively inexpensive and easy to ship by air. Demonstration of ≥36 hours of hypothermic storage with retention of heart function will be considered to be a successful demonstration of feasibility for progression to a Phase II SBIR proposal for further evaluation in large animal models and ex vivo human heart evaluation post-preservation.

在与约翰霍普金斯大学的布兰达彻教授的合作中，我们计划改进目前最常用的 心脏保存的临床方法，即0至+4 ° C的低温。我们将建立在我们的基础上 保存制剂Unisol™已被证明可将整个大型动物在低于+10 ° C的温度下保存6- 使用Unisol™进行全血液置换后8小时，恢复生理功能后功能正常 条件我们还表明，Unisol™可以在-7 ℃和-10 ℃下维持血管功能至少6天。 在Unisol™中在+4 ° C和+4 ° C储存18小时的小鼠心脏具有显著更快的恢复， 心脏功能优于保存在黄金标准低温心脏保存液Celsior（HTK）中的心脏。 受这些结果的鼓舞，我们建议对补充有靶向氧化试剂的Unisol进行评价， 细胞凋亡（应激耐受性增强剂）和代谢（代谢率抑制）在2个特定目标中， 人心肌细胞系体外选择最佳试剂浓度和异位心脏 移植模型，以评估在体外研究中开发的最佳补充制剂。率先 体外试验将使用alamarBlue，但将使用替代试验（包括锥虫）检查结局 蓝色、活/死染色和MTT测定。如果代谢活性显著丧失，则将评价细胞凋亡。 恢复到生理培养条件后1至2天观察。MALDI组织成像和蛋白质组学将 在选定的对照和表现最好的心脏上进行， 代谢物、脂质、酶产物和心脏切片中的翻译后修饰。体内 研究将评价来自体外研究的最佳制剂在低温储存7天内， 同系BALB/C小鼠，异位移植模型。对照组心脏将在高K+下保存 Unisol™（UHK）和HTK。我们预计，补充制剂将保持可行性， 心脏功能至少36小时，这是对目前所有练习方法的一个相当大的改进。基于 根据血管保存的初步数据，可以获得6-7天的心脏保存时间。这 创新不仅会增加存储时间，还将提供更紧密匹配的机会， 受体和潜在的耐受诱导。此外，我们正在改进最受欢迎的， 在临床实践中，真正的低温心脏储存方法相对便宜且简单 空运。将证明低温储存≥36小时并保留心脏功能 被认为是一个成功的示范可行性进展到第二阶段SBIR建议， 在大型动物模型中的进一步评价和离体人心脏保存后评价。